Molecular cloning (View forum version)



Chomolungma

Posted 12 June 2014 - 06:48 PM

Hi, can anyone tell me the  sequential steps involved, if you want to clone and molecular characterize a particular gene? Thanks 


bob1

Posted 12 June 2014 - 08:30 PM

Characterize in what manner - DNA sequence? or protein expression?


Chomolungma

Posted 13 June 2014 - 12:11 AM

I might need them both- I need to check and confirm for the presence of this rice gene in another unstudied rice variety, analyze the sequence features and clone it. Then want to study the expressd protein.... 


bob1

Posted 13 June 2014 - 01:13 AM

If the gene is conserved across a number of rice varieties or even across a number of species you can design consensus primers off the known sequence.  You would then use these to amplify your gene off a cDNA prep.  If you add restriction sites to the 5' end of your primers you can use these to clone the sequence, or you could use TA cloning.  The clones can then be sequenced.

 

From the sequenced region, you can then do 5' and 3' RACE to get the correct ends for the cDNA so as to give you a correct full length sequence.  Using the full sequence, design primers as before and clone the correct sequence.  Insert into an expression vector and work from there for protein characterization.  You can use the exact gene sequence to design primers for quantitative PCR for expression analysis.


Chomolungma

Posted 13 June 2014 - 02:05 AM

Thanks Thelymitra Pulchella....


bob1

Posted 13 June 2014 - 06:18 PM

My name on here is Bob (just as yours is Chomolungma - are you Tibetan by any chance?) - Thelymitra is the botanical name of the genus of flower I am using as an avatar...